Nitric oxide is a cell-signaling molecule involved in a number of physiological and pathophysiological processes. Modification of cysteine residues by nitric oxide, i.e. S- nitrosation, changes the function of a broad spectrum of proteins. This reaction represents an important post-translational modification that transduces nitric oxide- dependent signals. However, the detection and quantification of S-nitrosation in biological samples remains a challenge because of the lability of the S-nitrosation products: S-nitrosothiols (RSNOs). Our group recently developed a series of new reactions for RSNOs, which include bis-ligation, reductive ligation, and reductive elimination (to form dehydroalanine). These reactions selectively target on RSNOs and convert them to stable/detectable products. Based on control experiments, we hypothesize these reactions can be used to design new methods for the detection of RSNOs. In this project, we plan to pursue the following four Specific Aims: (1) to study bis-ligation based methods for detecting protein S-nitrosation; (2) to study reductive ligation based methods for detecting protein S-nitrosation; (3) to study dehydroalanine formation from S-nitrosocysteines and the applications in the detection of protein S- nitrosation; and (4) to validate the applications of new RSNO detection methods in biological systems. These studies will advance the understanding of the chemical and biological properties of RSNOs and permit studies of S-nitrosation-related nitric oxide signal transducation in complex systems.